JPS58146280A - Immobilized yeast having proliferation activity and its preparation - Google Patents

Abstract

PURPOSE:To prepare immobilized yeast having proliferation activity and remarkably strengthened activity, by proliferating the yeast at the surface of or in an immobilizing carrier. CONSTITUTION:A polymerizable monomer containing water or a non-polymerizable solvent is polymerized in the presence of light or ionizing radiation or a redox catalyst, etc. at or below room temperature to obtain a porous carrier having a pore size of >=5Angstrom . The carrier is sufficiently swollen with water, and stirred in a cultured liquid of the yeast to adsorb the yeast to the surface of the carrier. Thereafter, the carrier adsorbed with the yeast is immersed in a dilute solution of the polymerizable carrier monomer and the monomer is polymerized to form a polymer film on the surface of the carrier, or the carrier adsorbed with the yeast is further stirred in the culture liquid of the yeast.

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to an immobilized yeast product having proliferation ability and a method for producing the same. There is a lot of interest in producing ethanol by fermentation as a new energy source to replace petroleum. However, ethanol has been produced from sugar using fermentation ffi;
Since this is a batch reaction and the fermentation reaction rate is slow, there is now a need for continuous production and technology to speed up the reaction. In recent years, methods for immobilizing microorganisms including yeast have been investigated, and methods for immobilization using steam synthesis or whole natural polymeric substances as carriers have been proposed. The inventors of the present invention have also proposed a method for effectively immobilizing microorganisms and enzymes without significantly losing their original activity by mixing them with all monomers of microorganisms and enzymes and subjecting them to radioactive polymerization at low temperatures. Including the method proposed by the present inventors and others, the so-called comprehensive immobilization method (in C, all polymerizable monomers such as microorganisms and enzymes or mixed with natural polymers dissolved at elevated temperature) This is then solidified using means such as light or radiation irradiation, the use of a catalyst, or cooling to create an immobilized product.Therefore, in such conventional comprehensive methods, microorganisms and Enzymes are already present in the process of producing immobilization carriers and may be deactivated by the immobilization reaction.The purpose of the present invention is to eliminate the risk of such deactivation during yeast immobilization. It is an object of the present invention to provide an immobilized yeast having a proliferation ability that proliferates on the surface or inside of a carrier and whose number per unit volume is much higher than that at the time of identification, and whose activity is thereby significantly increased. The purpose of the present inventors is to provide a method for producing a yeast immobilized product.In order to achieve this objective, the inventors of the present application have made the following findings as a result of subjective research.
First, a porous carrier is prepared by various means in the absence of yeast, and this porous carrier is shaken in a nutrient solution containing all yeast to allow yeast to adsorb onto the surface of the whole porous carrier. one!
By continuing shaking, yeast can be immobilized while fully propagating, or a polymerizable monomer can be attached to the porous body adsorbed with yeast, which can be polymerized and immobilized, and then yeast can be cultured. We have developed a method for propagating yeast by shaking it in a solution, and the immobilized yeast having the ability to proliferate and a polymerizable monomer containing water or a non-polymerizable solvent are immobilized on the porous carrier of the present invention at room temperature. In the following steps, a porous carrier is prepared by polymerization, and the carrier is sufficiently swollen with water. After a while, the carrier is placed in a yeast culture solution to completely adsorb yeast on the surface of the carrier, and then a low concentration solution of the polymerizable carrier is prepared. We have arrived at a method for producing an immobilized yeast product with proliferation ability, which consists of immersing the single substance in water and polymerizing it to form a polymer film on the surface of the carrier, or shaking it in a yeast culture solution. In this way, the method of the present invention involves first producing the entire immobilization carrier through various chemical reactions in the absence of yeast, and then polymerizing a small amount of polymerizable monomer through various chemical reactions in the presence of yeast. or immobilize without using polymerizable monomers or chemical reactions. Therefore, the method of the present invention has the advantage that yeast is less likely to be inactivated than the conventional inclusion method in which yeast is already present when the carrier is prepared and the yeast is likely to be inactivated by the reaction. Furthermore, unlike most conventional methods that utilize only the yeast present at the time of immobilization, the present invention allows yeast to grow on the surface or inside the immobilized material, increasing the number of yeast per unit volume at the time of immobilization. It has the characteristic of significantly increasing the activity of the immobilized product. In the present invention, appropriate flexibility of the porous carrier is indispensable for the adsorbed yeast to actively proliferate and for the carrier to have the receptivity to accept the increase in yeast volume due to increase in yeast M. This is achieved by selecting the material for making the carrier and soaking the produced porous carrier in water for a long period of time to allow it to swell sufficiently.The pore size of a normal porous carrier is limited by the size of the yeast. It is desirable that the polymerizable monomer is 5 to 5.Next, the method of the present invention will be explained in detail.First, the polymerizable monomer used in the method of the present invention belongs to the following [A1 group]. One or more monomers that do not crystallize and are in a stable supercooled state even at room temperature to low temperature, or
Although it does not have supercooling property itself, it has 30
% or less, the monomers of the CB group maintain supercooling properties as a whole when mixed. [A] Group monomers: hydroxybutyl acrylate, hydroquine ethyl acrylate, hydroxypropyl methacrylate, hydroxyzolopyl acrylate, hydroxybutyl methacrylate, hydroxybutyl acrylate, hydroxypentyl methacrylate, hydroxypentyl acrylate, hydroxyhexyl methacrylate, hydroxyhexyl Acrylate, hydroxyhebutyl methacrylate, hydroxyhebutyl acrylate, glycidyl methacrylate, glycidyl acrylate, diethylene glycol dimethacrylate, diethylene gelicol diacrylate, tetraethylene glycol diacrylate,
Tetraethylene glycol diacrylate, polyethylene glycol diacrylate, polyethylene glycol diacrylate, ethylene glycol dimethacrylate, ethylene glycol diacrylate, propylene glycol dimethacrylate, propylene glycol diacrylate, dipropylene glycol dimethacrylate,
Dipropylene glycol) Schifkuri L/-t Volip°'v7 Gly-'' dimethacrylate, polypropylene glycol diacrylate, butylene glycol dimethacrylate. Butylene glycol diacrylate, betanediol dimethacrylate, betanediol diacrylate,
hexanediol dimethacrylate, hexanediol diacrylate, heptanediol dimethacrylate,
heptanediol diacrylate, neopentyl glycol dimethacrylate, neopentyl glycol diacrylate, trimethylolethane trimethacrylate,
Trimethylolethane triacrylate, trimethylolzoronotrimethacrylate, trimethylolprobant/acrylate, glycerol monomethacrylate, glycerol monoacrylate, glycerol dimethacrylate, glycerol diacrylate, glycerol trimethacrylate, glycerol triacrylate, Diethylaminoethyl methacrylate, diethylaminoethyl acrylate, diethylamine butyl methacrylate, diethylaminobutyl acrylate, dimethylaminoethyl methacrylate, dimethylaminoethyl acrylate, dimethylamine butyl methacrylate, dimethylaminobutyl acrylate, benzyl methacrylate, methoxypolyethylene glycol methacrylate, methoxypolyethylene glycol acrylate,
Ethoxypolyethylene glycol methacrylate, ethoxypolyethylene glycol acrylate, methoxypropylene glycol methacrylate, methoxypropylene glycol acrylate, ethoxypropylene glycol methacrylate, ethoxypropylene glycol acrylate, etc. [B] Group monomer: vinyl acetate, vinyl propionate, vinyl butyrate, methyl methacrylate, methyl acrylate, ethyl methacrylate, ethyl acrylate, propyl methacrylate, propyl acrylate, butyl methacrylate, butyl acrylate, pentyl methacrylate, pentyl acrylate, hexyl methacrylate , hexyl acrylate, lauryl methacrylate, lauryl acrylate, stearyl methacrylate, stearyl acrylate, styrene, vinyltoluene, sulfonated styrene, acrylic acid, methacrylic acid, aminostyrene, vinylpyrrolidone, acrylamide, methacrylamide, methylenebisacrylamide, methylolacrylamide,
Acrylonitrile, methacrylaterile, sialyl lunthalate, itaconic acid, maleic anhydride, diallyl inphthalate, diallyl succinate, diallyl itaconate, divinylbenzene, triallyl cyanurate, etc. Next, the non-polymerizable properties a used in the present invention are the following [group 03 solvents:

[0] Group: Acetone, methyl ethyl ketone, ethylene glycol diethyl ether, methyl formate, ethyl formate, methyl acetate, glycol carbonate, ethyl acetate, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether acetate, glycol diacetate, Glycol monomethyl ether, glycol monoethyl ether, glycol monobutyl ether, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, methyl alcohol, ethyl alcohol, n, /J
propyl alcohol, inpropyl alcohol, ethylene glycol, 5ec-methyl alcohol, etc. [The polymerizable monomers shown in the A3 group and [E] group are mixed with water or the non-polymerizable solvent shown in the [0] group, and the mixture is brought to room temperature or below, and a polymerization reaction is carried out by irradiation with photo-heavy ionizing radiation. A porous polymeric carrier is made by Next, it is preferable to immerse this porous carrier in water for several days, swell and swell it thoroughly with water, and then sterilize it using an autoclave or the like. (1) By shaking the thus prepared porous carrier in a whole yeast culture solution for 24 hours, yeast is adsorbed onto the surface of the porous carrier. This yeast-adsorbed porous carrier is immersed in a low concentration solution of a polymerizable monomer for a short time, and a thin polymer film is formed on the carrier surface through photo or ionizing radiation polymerization, thereby achieving immobilization. . By shaking this immobilized whole yeast culture solution to maintain conditions suitable for aerobic yeast growth, an immobilized growth 114 mother is obtained. (2) By incubating the porous carrier prepared as described above in a yeast culture solution, yeast is adsorbed on the surface of the porous carrier. The porous carrier on which the yeast has been adsorbed is left as it is for fermentation. Invades the inside of the carrier.Through the above process, the yeast is immobilized on the surface and inside of the porous carrier while maintaining an active growth state. Therefore, it is essential that the porous carrier has a suitable degree of flexibility, and due to this flexibility, the walls of the pores inside the carrier undergo appropriate Qτ deformation, which prevents the increase in yeast volume due to yeast proliferation. The temperature employed as the polymerization reaction temperature in carrying out the entire method of the present invention (is a room temperature of 25°C to -196°C).
It is used after being cultured in a preculture solution containing ~5 sugars, peptone, yeast oil field liquor, and salt. In the method of the invention, the radiation sources employed are light from low- or high-pressure mercury lamps, X-rays, gamma rays, beta rays, electron beams, alpha rays, mixed radiation from chemical reactors, spent fuel, fission products, The selection of the irradiation dose is relatively free since the yeast is likely to be irradiated with light or radiation during the irradiation to produce the porous carrier, but the choice of the irradiation dose is relatively free. at least 50 monomers
Irradiation is performed at an amount of IX]O'R or more necessary to cause hardness of % or more. In the case of radiation, IX], 03-109 at the dose rate], X], O') (, ~], X] O' 1
(It is desirable that the irradiation dose be I/-1°. If the irradiation is too large after the yeast has been completely adsorbed onto the porous carrier prepared in this way, a thick dose Gτ will have a negative effect on the yeast O, so it is desirable that it be as small as possible. However, in order to cure the polymerizable monomer by 50% or more, irradiation of 1X10'R or more is necessary.Therefore, in the case of radiation, 1X10' to IX], 09) at a dose rate of t/14. X], O”~5X], 06R1 preferably 5
．． A radiation dose of X10' to IXI, Q'R is required. Instead of polymerization by irradiation with light or ionizing radiation in the method of the present invention, redox catalysts or other catalysts can be used to carry out the polymerization. However, in the case of irradiation polymerization using light or ionizing radiation, there is no possibility that catalyst residues and the like remain in the immobilized material as in the case of catalytic polymerization, and yeast growth can be carried out more favorably. In the case of irradiation polymerization, polymerization can be easily carried out in a short time, and furthermore, various properties can be changed over a wide range by combining various monomers. The present invention will be explained in more detail below using Examples. Ethanol produced in the examples was quantified using alcohol dehydrogenase. Example 1 50 ml of water was added to 2.5 grams of methoxypolyethylene glycol methacrylate (M-23G) as a monomer, and after thorough mixing, the gamma rays from Co were irradiated at -78°C. The entire porous carrier was irradiated, cut into small pieces, and then shaken in water for 4 days to fully swell in water. Yeast were precultured under aerobic conditions at 30°C for 24 hours in a preculture medium containing 1% glucose, 0.1% molasses, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract. , 1 g of this yeast pre-culture solution is exposed to glucose for 12 times, ? Pre-molasses, 0.15 yeast extract, 0.25% NH
<OL, 0.5% 2HPO4, O-02
5%Mg-8O4,7H20,0,1%N'aO/1.
, mixed with 20d of complete medium containing 0.001% Oa OL2 and 0.3% lactic acid. The porous carrier swollen in water was placed in a complete medium containing whole yeast, and aerobic conditions were maintained for 24 hours. After yeast attachment, the entire adsorbed porous carrier was taken out, immersed in a solution of 5% M-230i dissolved in the preculture medium for 1 minute, taken out again, and exposed to γ-rays from 60Co under aseptic conditions at room temperature. Yeast was identified as a carrier by irradiation with 'e5X10''+. The immobilized product was immersed in complete medium and cultured at 30°C under aerobic conditions.24
The carriers were taken out and replaced with fresh complete medium every hour. After maintaining aerobic conditions for a certain period of time, part of the immobilized product is shaken with a complete medium in an equal amount to the entire immobilized product (C), and all of the ethanol is produced by a fermentation reaction in the first hour. The alcohol content was used as an indicator of the activity of the immobilized product.A mixture of a complete medium suspension of non-immobilized yeast and the same volume of complete medium obtained in exactly the same manner except that an immobilization carrier was added. Therefore, the activity of the immobilized product was measured in the same way as the immobilized product.The results are all compared and shown in Figure 1.As the culture continued under aerobic conditions, the activity of the immobilized product (curve A) rapidly increased, indicating that the immobilized product The activity reached 10 times that of untreated yeast (curve B) and maintained its high activity for more than 20 days.Example 2 Using 2-hydroquine ethyl acrylate (1-I
EA, )5me and methoxypolyethylene glycol methacrylate (M23G)5m12VC
After mixing thoroughly, -78
A porous carrier was prepared by irradiating with gamma rays from 6000°C to 1 x 10°R5.The yeast was shredded into small pieces and then shaken in water for 4 days to thoroughly water the yeast. under aerobic conditions, 3
It was precultured at 0°C for 24 hours in the same mJ culture medium as in Example 1, and mixed with 20 g of the same complete medium as in Example 1. The porous carrier swollen in water was placed in a complete medium C containing yeast, and aerobic conditions were maintained by shaking. After maintaining aerobic conditions for a certain period of time, a portion of Fixation 1 was gently stirred with an equal volume of complete medium to allow complete ethanol production by fermentation reaction, and the first 1
The amount of alcohol produced over time was used as a guide to the activity of fixed substances. The activity was also measured in the same manner as with the immobilized product using a mixture of a small amount of a complete medium of non-immobilized yeast eggplant suspension obtained by the same procedure except that an immobilized carrier was added, and the same volume of the complete medium. the result? Compare with Fig. 2] - is shown. As the culture continued under aerobic conditions, the activity of the immobilized product (curve A) rapidly increased, reaching 13 times the activity of unimmobilized yeast (curve B), and maintained this high activity for more than 20 days. . Furthermore, FIG. 3 shows the results of fermentation reaction using an immobilized substance, which was allowed to continue for one hour until all the sugars were converted to ethanol. 100% ethanol yield is obtained in 150 minutes for the immobilized product (curve aA), but for undefined yeast (curve B) within the same time period.
), an ethanol yield of only 10% was obtained, and much higher activity was obtained in immobilized I7 yeast compared to such yeast without immobilized I7.

[Brief explanation of the drawing]

Figure 1 is a graph comparing the activity of the yeast immobilized product obtained in Example 1 with the activity of unimmobilized yeast. FIGS. 2 and 3 are graphs comparing the activity of the yeast immobilized product obtained in Example 1112 with the activity of unimmobilized yeast. Figures 1 and 2 (in the horizontal axis is culture time (hr))
+ The vertical axis is alcohol concentration (%9). In Figure 3, the horizontal axis is fermentation time (minutes) and the vertical axis is alcohol yield (%). Patent applicant Japan Atomic Energy Research Institute

Claims (1)

[Scope of Claims] 1. An immobilized yeast having growth ability immobilized on a porous carrier. 2. The first porous carrier has a pore size of 5'A or more.
Yeast immobilization of the term. 3 A polymerizable monomer containing water or a non-polymerizable solvent is polymerized at room temperature or below to prepare a porous carrier, and after sufficiently swelling the carrier with water, it is shaken in a yeast culture solution to form a porous carrier. It consists of adsorbing yeast on the surface, and then immersing it in a low concentration bath solution of a polymerizable simple substance to polymerize the simple substance to form a complete polymer film on the surface of the carrier, or breaking it as it is and shaking it in a yeast culture solution. A method for producing an immobilized yeast product having proliferation ability. 4. The method of item 3, wherein the polymerization 1 is carried out by irradiation with light or ionizing radiation. 5. In the case of polymerization by irradiation with ionizing radiation, the amount of irradiation per gland is lX103~lXl091'/off1J amount rate TeIx104~time1X1.071<, and the irradiation amount for polymerization after yeast adsorption is iMi is ]Xl, 0' ~ lXl09R/h (D
% flJ H rate], X10'~5X]06) The method of paragraph 6.